SEARCH · PubMed Health

Results for “NtrC”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed1988

The effect on the function of the transcriptional activator NtrC from Klebsiella pneumoniae of mutations in the DNA-recognition helix.

We have constructed mutations in what we predict to be the DNA-recognition helix of Klebsiella pneumoniae NtrC, which regulates transcription from promoters under global nitrogen control. Mutations which disrupt the helix lead to complete loss of function. All point mutants tested were able to activate transcription from the sigma 54-dependent glnA promoter, but only those retaining some ability to recognise NtrC binding sites, as evidenced by their ability to repress the ntrB promoter and the upstream glnA promoter, were able to activate the nifL promoter. One mutant, which contained an amino acid substitution in the turn of the DNA-binding motif as well as in the recognition helix, suppressed mutations in the NtrC binding sites upstream from the nifL promoter, but only if both sites bore equivalent transitions. This confirms that the DNA-binding motif for this class of transcriptional activator has been correctly identified and suggests that binding of NtrC can be cooperative.

Amino Acid Sequence↗

PubMed1998

DNA binding and oligomerization of NtrC studied by fluorescence anisotropy and fluorescence correlation spectroscopy.

Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding sites. The following results were obtained: (i) the dissociation constant K d for binding of one NtrC dimer to a single binding site was the same for the wild-type and mutant proteins within the error of measurement. (ii) The value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to 5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the dissociation constant we calculated that two ion pairs form upon binding of one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA duplex with two binding sites occured with essentially no cooperativity. Titration curves of NtrCS160Fbinding to the same duplex demonstrated that more than two protein dimers of the mutant protein could bind to the DNA.

Amino Acid Sequence↗

PubMed1991

Substitutions at a single amino acid residue in the nitrogen-regulated activator protein NTRC differentially influence its activity in response to phosphorylation.

Four substitutions at serine residue 160 which increase the activity of the sigma 54-dependent activator protein NTRC in the absence of NTRB have been analysed in detail. Mutagenesis of the putative phosphoacceptor site of NTRC and analysis of double mutants indicate that the positive control function of the S160W and S160C mutants is phosphorylation-dependent, whereas the activity of the S160Y and S160F mutants is phosphorylation-independent. This was confirmed with two purified mutant proteins in vitro. Occupancy of tandem NTRC-binding sites upstream of the Klebsiella pneumoniae nifL promoter by S160W protein is also phosphorylation-dependent in contrast to occupancy by S160F protein, confirming that both the DNA-binding and activator functions of NTRC are influenced by phosphorylation. The S160W and S160C mutants are apparently more responsive than wild-type protein to 'cross-talk' by other members of the histidine protein kinase family but are less responsive to phosphorylation and dephosphorylation mediated by NTRB.

Amino Acid Sequence↗

PubMed2000

The ntrB and ntrC genes are involved in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 and its ntrA (rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the ntrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC and ntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and ntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that the ntrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilense Sp7.

Ammonia↗

PubMed1991

Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription.

Most strains of Pseudomonas aeruginosa isolated from the respiratory tracts of cystic fibrosis patients have a mucoid colony morphology due to the synthesis of an expolysaccharide called alginate. The algB gene product (AlgB) is necessary for the high-level production of alginate in mucoid P. aeruginosa. In this study, AlgB was shown to be involved in the transcription of algD, a gene previously demonstrated to be activated in mucoid P. aeruginosa. In vitro and in vivo expression studies reveal that algB encodes a protein with a molecular size of 49 kDa. The DNA sequence of a 2.2-kb P. aeruginosa fragment containing algB was also determined. The amino-terminal domain of AlgB was found to be conserved with the amino-terminal domains of the response regulator class of two-component regulatory proteins. The central domain of AlgB has sequences highly conserved with those in the NtrC subfamily of transcriptional activators (NtrC, NifA, HydG, DctD, FlbD, TyrR, and PgtA). The central domain of AlgB also contains a potential nucleotide binding site. AlgB is the first NtrC homolog described from P. aeruginosa. At the carboxy terminus of AlgB, a helix-turn-helix motif was observed, suggesting that AlgB is a DNA-binding protein. The strongly conserved NtrC-like central domain of AlgB is not present in AlgR, another alginate response regulator. This study therefore identifies and characterizes the second of at least two unique response regulators used by P. aeruginosa to control alginate gene expression.

Alginates↗

PubMed1993

Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele.

In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three sigma 54 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdctA), respectively. Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters. The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed. The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283. Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80% of the wild-type level. Thus, the nifA gene is dispensable for symbiotic N2 fixation.

Alleles↗

PubMed1999

MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein.

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma(54)-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent K(d) of unphosphorylated NtrC for MgATPgammaS is 100 microM at 25 degrees C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 microM at 37 degrees C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of sigma(54)-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21(ras) and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn(2+) binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.

Adenosine Triphosphate↗

PubMed1995

The ntrBC genes of Azospirillum brasilense are part of a nifR3-like-ntrB-ntrC operon and are negatively regulated.

A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.

Amino Acid Sequence↗

PubMed2003

Reconstitution and characterization of NtrC protein in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12.

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.

Bacterial Proteins↗

PubMed1985

Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus binding sequence.

We have investigated the DNA-binding ability of a nitrogen regulatory protein, the product of the ntrC gene, to several nitrogen-regulated promoters in Salmonella typhimurium. The ntrC protein is able to bind to the regulatory region (dhuA) of an operon coding for genes involved in the active transport of histidine, but not to another transport-related regulatory region, argTr. It bound to two different sites within the regulatory region of glnA (glutamine synthetase) and to one site in the regulatory region for the ntrBC operon. A consensus sequence has been derived from these four binding sites. The binding sequence displays dyad symmetry, as expected for the dimeric ntrC protein. The relationship of the binding sites to regulation of transcription initiation and termination, and to published homologies within the sequences of regulatory sites for nif genes is discussed.

Amino Acid Transport Systems↗

PubMed1986

Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins.

We have determined the nucleotide sequences of two genes from Klebsiella pneumoniae, nifA, the nif-specific activator of transcription and ntrC, the bifunctional regulatory protein involved in 'nitrogen control'. These sequences differ significantly from those previously published. In particular, nifA extends 40 codons beyond the stop codon reported earlier. This extension encodes a putative DNA-binding domain strongly homologous to the Rhizobium meliloti nifA protein and to some extent to the ntrC protein. In all three proteins this domain is linked by a segment of variable length to a strongly conserved central domain of 240 residues. A short segment having the properties of an interdomain linker joins the central region to an N-terminal domain, which is weakly related in the case of the two nifA proteins. This homology is not shared by the N-terminal domain of ntrC, which is clearly but unexpectedly related to the N-terminal domains of a diverse set of procaryotic pleiotropic control proteins, including ompR, dye and nusA from Escherichia coli and spoOA and spoOF from Bacillus subtilis.

Amino Acid Sequence↗

PubMed1995

A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules.

During signal transduction, response regulators of two-component systems are phosphorylated in a conserved receiver module. Phosphorylation induces activation of the non-conserved output domain. We fused various domains of the response regulators NtrC, PhoB or CheB to the DNA binding domain of lambda repressor. Analysis of these hybrid proteins shows that the receiver modules of NtrC and PhoB are potential dimerization domains. In the unphosphorylated proteins, the ability of the receiver modules to dimerize is masked due to inhibition by their output domains. Inhibition can be relieved in two ways: phosphorylation of the receiver module or deletion of the output domain. In contrast, the receiver module of CheB lacks this ability for dimerization. We propose a model which groups response regulators into two classes. Common to both classes is the interaction between receiver and output domain in the unphosphorylated protein. In class I (e.g. NtrC and PhoB), this interaction leads to the inhibition of the receiver module. Phosphorylation relieves inhibition, thereby inducing activation via dimerization of the receiver modules. In class II (e.g. CheB), the interaction between receiver and output domain results in inhibition of the output domain. Phosphorylation relieves inhibition, thereby activating the output domain.

Adenosine Triphosphatases↗

PubMed1999

Solution structure of the DNA-binding domain of NtrC with three alanine substitutions.

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.

Alanine↗

PubMed1999

Investigation into the role of the response regulator NtrC in the metabolism and virulence of Brucella suis.

During infection, Brucella species have to adapt to a range of different environments. Environmental sensing in bacteria often involves the concerted action of two-component regulatory systems consisting of sensor and response regulator components. In this study, we identified, cloned and sequenced four independent response regulator gene fragments from Brucella melitensis. One amplified gene fragment showed nearly 90% identity to the response regulator subfamily of NtrC transcriptional activators, and further analysis revealed the presence of an adjacent gene encoding the sensor protein NtrB. The NtrBC two-component regulatory system has been shown to play varying roles in nitrogen metabolism and potentially in virulence in other bacterial species. A B. suis ntrC isogenic mutant was constructed which showed no significant differences in growth rates compared to the wild-type strain when grown at different temperatures in vitro. However, the mutant exhibited a reduction in metabolic activity in the presence of many amino acids. The mutation did not affect survival or multiplication of B. suis in macrophages, but during the initial stages of infection in the murine brucellosis model, the ntrC mutant showed a reduced ability to multiply rapidly in splenic tissue.

Amino Acid Sequence↗

PubMed1994

The role of regulatory genes nifA, vnfA, anfA, nfrX, ntrC, and rpoN in expression of genes encoding the three nitrogenases of Azotobacter vinelandii.

Several regulatory gene mutants of Azotobacter vinelandii were tested for ability to synthesize functional nitrogenase-1 (Nif phenotype), nitrogenase-2 (Vnf), or nitrogenase-3 (Anf). While nifA mutants were Nif-, Vnf+, and Anf+/-, and ntrC mutants were Nif+, Vnf+, and Anf+, nifA ntrC double mutants were Nif-, Vnf-, and Anf-. A vnfA mutant was Nif+, Vnf+/-, and Anf+/-, and an anfA strain was Nif+, Vnf+, and Anf-. lacZ fusions in the nifH, vnfH, vnfD, anfH, and nifM genes of Azotobacter vinelandii were constructed and introduced into wild-type and regulatory mutants of A. vinelandii. Expression of these operons correlated with the growth phenotype of the regulatory mutants. Apparently either NifA or NtrC can activate expression of nifM. Also, expression of the anf operon required the NifA transcriptional activator, although there are no NifA binding sites at appropriate locations upstream of anfH (or anfA). The results confirm previous reports that VnfA and AnfA are required for expression of vnf and anf genes, respectively, and that VnfA is involved in repression of the nifHDK operon in the absence of molybdenum and of the anfHDGK operon in the presence of vanadium.

Amino Acid Sequence↗

PubMed1999

Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry.

We have developed a mass spectrometry based method for the identification of linker regions and domain borders in multidomain proteins. This approach combines limited proteolysis and in-gel proteolytic digestions and was applied to the determination of linkers in the transcription factor NtrC from Escherichia coli. Limited proteolysis of NtrC with thermolysin and papain revealed that initial digestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domain and the still covalently linked OC-domains. Subsequent steps in limited proteolysis afforded further cleavage of the OC-fragment into the O- and the C-domain at accessible amino acid residues. Mass spectrometric identification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker between R- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement with papain and subtilisin digestion. The region between amino acid residues Thr-389 and Gln-396 marked the hitherto unknown linker sequence that connects the O- with the C-domain. High abundances of proline-, alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of related NtrC response regulator proteins. While R- and C-domains remained stable under the applied limited proteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised the sequence from Ile-140 to Arg-320. ATPase activity was lost after separation of the R-domain from the OC-fragment. However, binding of OC- and C- fragments to specific DNA was observed by characteristic band-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain. The outlined strategy proved to be highly efficient and afforded lead information of tertiary structural features necessary for protein design and engineering and for structure-function studies.

Amino Acid Sequence↗

PubMed1990

FlbD of Caulobacter crescentus is a homologue of the NtrC (NRI) protein and activates sigma 54-dependent flagellar gene promoters.

The periodic transcription of flagellar genes in the Caulobacter crescentus cell cycle is controlled, in part, by their organization in a regulatory hierarchy. The flbG (hook operon), flaN, and flagellin gene operons, which are at the lowest levels of the hierarchy and expressed late in the cell cycle, contain Ntr-like promoters. We report that flbD, one of the early genes required in trans for expression of these operons, codes for a 52-kDa protein homologous to the transcriptional activators NtrC (NRI), NifA, DctD, HydG, and XylR. Our results show that in Escherichia coli flbD partially complements glnG (ntrC) mutations and stimulates transcription of the C. crescentus sigma 54 RNA polymerase-dependent flbG gene. Additionally, the sequence predicts that FlbD protein, along with NtrC, DctD, and HydG proteins, is structurally related at the amino-terminal domain to a larger family of response regulators that mediate cellular responses to environmental stimuli. FlbD may be a singular member of this large protein family in that its function is tied to an internal cell-cycle signal. FlbD is also unusual in that its amino-terminal domain contains only one of the three residues conserved in previously described members of this family of response regulators.

Amino Acid Sequence↗

PubMed2000

Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element.

The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a -12/-24 sigma(N)-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5' end to the RNA was identified at position 641, 12 bp downstream from the -12/-24 promoter. Footprinting experiments showed that the G residues at positions -26 and -9 are hypermethylated, and that the region from -10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the sigma(N)-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

Bacterial Proteins↗